Immunochemical detection of in vivo advanced glycosylation endproducts

ABSTRACT

The circulating advanced glycosylation endproducts Hb-AGE, serum AGE-peptides and urinary AGE-peptides are disclosed as long term markers of diseases and dysfunctions having as a characteristic the presence of a measurable difference in AGE concentration. Diagnostic and therapeutic protocols taking advantage of the characteristics of these AGEs are disclosed. Antibodies which recognize and bind to in vivo-derived advanced glycosylation endproducts are also disclosed. Methods of using these antibodies as well as pharmaceutical compositions are also disclosed, along with numerous diagnostic applications, including methods for the measurement of the presence and amount of advanced glycosylation endproducts in both plants and animals, including humans, as well as in cultivated and systhesized protein material for therapeutic use.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a a Continuation-In-Part of copendingapplication Ser. No. 07/956,849, filed Oct. 1, 1992, pending, which is aContinuation-In-Part of application Ser. No. 07/811,579, filed Dec. 20,1991, now abandoned, by the inventor herein. Priority under 35 U.S.C.§120 is claimed as to the above earlier filed Applications, and thedisclosures thereof are incorporated herein by reference in theirentireties.

RELATED PUBLICATIONS

The following articles are noted as they are generally directed to thesubject matter of the present invention: "FUNCTION OF MACROPHAGERECEPTOR FOR NONENZYMATICALLY GLYCOSYLATED PROTEINS IS MODULATED BYINSULIN LEVELS", Vlassara, Brownlee and Cerami, DIABETES (1986), Vol. 35Supp. 1, Page 13a; "ACCUMULATION OF DIABETIC RAT PERIPHERAL NERVE MYELINBY MACROPHAGES INCREASES WITH THE PRESENCE OF ADVANCED GLYCOSYLATIONENDPRODUCTS", Vlassara, H., Brownlee, M., and Cerami, A. J. EXP. MED.(1984), Vol. 160, pp. 197-207; "RECOGNITION AND UPTAKE OF HUMAN DIABETICPERIPHERAL NERVE MYELIN BY MACROPHAGES", Vlassara, H., Brownlee, M., andCerami, A. DIABETES (1985), Vol. 34, No. 6, pp. 553-557;"HIGH-AFFINITY-RECEPTOR-MEDIATED UPTAKE AND DEGRADATION OFGLUCOSE-MODIFIED PROTEINS: A POTENTIAL MECHANISM FOR THE REMOVAL OFSENESCENT MACROMOLECULES", Vlassara H., Brownlee, M., and Cerami, A.,PROC. NATL. ACAD. SCI. U.S.A. (September 1985), Vol. 82, pp. 5588-5592;"NOVEL MACROPHAGE RECEPTOR FOR GLUCOSE-MODIFIED PROTEINS IS DISTINCTFROM PREVIOUSLY DESCRIBED SCAVENGER RECEPTORS", Vlassara, H., Brownlee,M., and Cerami, A. JOUR. EXP. MED. (1986), Vol. 164, pp. 1301-1309;"ROLE OF NONENZYMATIC GLYCOSYLATION IN ATHEROGENESIS", Cerami, A.,Vlassara, H., and Brownlee, M., JOURNAL OF CELLULAR BIOCHEMISTRY (1986),Vol. 30, pp. 111-120; "CHARACTERIZATION OF A SOLUBILIZED CELL SURFACEBINDING PROTEIN ON MACROPHAGES SPECIFIC FOR PROTEINS MODIFIEDNONENZYMATICALLY BY ADVANCED GLYCOSYLATION END PRODUCTS", Radoff, S.,Vlassara, H. and Cerami, A., ARCH. BIOCHEM. BIOPHYS. (1988), Vol. 263,No. 2, pp. 418-423; "ISOLATION OF A SURFACE BINDING PROTEIN SPECIFIC FORADVANCED GLYCOSYLATION ENDPRODUCTS FROM THE MURINE MACROPHAGE-DERIVEDCELL LINE RAW 264.7", Radoff, S., Vlassara, H., and Cerami, A.,DIABETES, (1990), Vol. 39, pp. 1510-1518; "TWO NOVEL RAT LIVER MEMBRANEPROTEINS THAT BIND ADVANCED GLYCOSYLATION ENDPRODUCTS: RELATIONSHIP TOMACROPHAGE RECEPTOR FOR GLUCOSE-MODIFIED PROTEINS", Yang, Z., Makita,Z., Horii, Yo, Brunelle, S., Cerami, A., Sehajpal, P., Suthanthiran, M.and Vlassara, H., J. EXP. MED., Vol. 174, pp. 515-524; "HUMAN AND RATMESANGIAL CELL RECEPTORS FOR GLUCOSE-MODIFIED PROTEINS: POTENTIAL ROLEIN KIDNEY TISSUE REMODELLING AND DIABETIC NEPHROPATHY", Skolnik, E.,Yang, Z., Makita, Z., Radoff, S., Kirstein, M., and Vlassara, H., J.EXP. MED., Vol. 174, pp. 931-939; and "HEMOGLOBIN-AGE: A CIRCULATINGMARKER OF ADVANCED GLYCOSYLATION", Makita, Z., Vlassara, H., Rayfield,E., Cartwright, K., Friedman, E., Rodby, R., Cerami, A., and Bucala, R.,SCIENCE, (In Press). All of the foregoing publications and all otherreferences cited herein are incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to the detection and measurementof nonenzymatically glycosylated proteins, and particularly to methodsand associated materials for the detection and measurement of proteinsthat have been nonenzymatically glycosylated in vivo.

Reducing sugars, e.g., glucose, have been shown to reactnon-enzymatically with protein amino groups to form a diverse series ofprotein bound moieties with fluorescent and crosslinking properties.These compounds, called advanced glycosylation endproducts ("AGEs"),have been implicated in the structural and functional alteration ofproteins during aging and in certain diseases, e.g., long-term diabetes.Several AGEs have been identified on the basis of de novo synthesis andtissue isolation procedures.

The reaction between reducing sugars and the free amino groups ofproteins initiates the post-translational modification process calledadvanced glycosylation. This process begins with a reversible reactionbetween the reducing sugar and the amino group to form a Schiff base,which proceeds to form a covalently-bonded Amadori rearrangementproduct. Once formed, the Amadori product undergoes furtherrearrangement to produce AGEs.

Because these reactions occur slowly, proteins may accumulatesignificant amounts of Amadori products before accumulating a measurableamount of AGEs in vivo. These AGEs can cause protein crosslinking, whichin turn may reduce the structural and/or functional integrity of organsand organ parts, thus ultimately reducing or impairing organ function.

The advanced glycosylation process is particularly noteworthy in that itoccurs in proteins with long half-lives, such as collagen and underconditions of relatively high sugar concentration, such as in diabetesmellitus. Numerous studies have suggested that AGEs play an importantrole in the structural and functional alteration which occurs inproteins during aging and in chronic disease.

Additionally, advanced glycosylation endproducts are noted to form morerapidly in diabetic, galactosemic and other diseased tissue than innormal tissue.

Certain advanced glycosylation endproducts are believed to have incommon a characteristic yellow-brown pigmentation, a characteristicfluorescence spectrum and the ability to form protein-proteincrosslinks. AGEs form in vivo and have been isolated from naturallyglycosylated material. These products are present in low abundance, arestructurally heterogeneous and are labile to chemical reduction andhydrolysis. De novo synthesis and isolation procedures have led to theidentification of several AGEs, such as2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole ("FFI");5-hydroxymethyl-1-alkylpyrrole-2-carbaldehyde ("Pyrraline");1-alkyl-2-formyl-3,4-diglycosyl pyrrole ("AFGP"), a non-fluorescentmodel AGE; carboxymethyllysine; and pentosidine. However, the in vivoformation of AGEs is not limited to these precise chemical compounds,and newly discovered AGEs are addressed herein.

The study of specific AGEs synthesized in vitro in the past hasnecessitated the use of chemical reduction and hydrolysis procedures.This has left open the possibility that naturally occurring AGEs wouldinclude other compounds with alternative structures which differ fromthe model compounds which have been isolated.

Efforts have also been made to develop antibodies to in vivo AGEs,however no instances of success are known or have been reported. Thus,Nakayama et al., BIOCHEM. BIOPHYS. RES. COMM., 162:2, pp. 740-745 (1989)studied protein bound AGEs and in particular, raised antisera againstAGE-KLH derived from in vitro glycosylation. These antisera exhibitedhigh affinity binding, and the serial dilution curves of in vitro-formedAGE-BSA, AGE-HSA and AGE-RNAse A were noted to parallel each other,suggesting that a structure in common among these particularAGE-proteins is recognized by the antisera. Further study to determinewhether the structure recognized stems from advanced Maillard reactionsor from the early-stage compounds, such as Schiff base adducts andAmadori rearrangement products were conducted using a number of reducingagents. Treatment with a reducing agent did not decreaseimmunoreactivity, and FFI was not recognized by the antibodies.Importantly, the antibodies prepared and tested by Nakayama et al. werenot determined to react with AGEs formed in vivo.

Horiuchi et al., J. BIOL. CHEM., 266(12), pp. 7329-7332 (1991) preparedpolyclonal and monoclonal antibodies against in vitro-derived AGE-bovineserum albumin. The Horiuchi et al. antibodies also recognized invitro-derived AGE-human serum albumin and AGE-hemoglobin, but did notrecognize unmodified counterparts. Treatment of these AGE proteins witha reducing agent had no effect on immunoreactivity. Like the antibodiesof Nakayama et al., the antibodies prepared by Horiuchi et al. were notdetermined to react with in vivo-formed AGEs.

Accordingly, despite the facility with which antibodies have beenprepared in the art, the reactivity of such antibodies with invivo-formed AGEs has not been previously achieved. The preparation ofsuch antibodies is desirable as it makes possible the development andimplementation of diagnostic and therapeutic protocols addressing theformation of advanced glycosylation endproducts in mammals includinghumans.

In this context, parent application Ser. No. 07/811,579, now abandoned,discloses the preparation of an antiserum that contains antibodiesreactive with in vivo-formed advanced glycosylation endproducts. Amongthe advanced glycosylation endproducts against which antibodies wereraised, the reaction product of hemoglobin and a reducing sugar (Hb-AGE)was included. In addition, data were presented that compared this AGEfavorably with HbA_(1c) in terms of its use as a diagnostic agent.

The present application seeks to present further data cumulative on theactivity of Hb-AGE, thereby emphasizing its expanded capabilities. Also,the role of serum- and urinary AGE peptides as markers of disease anddysfunction is further elaborated herein.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, the advanced glycosylationendproduct involving a complex with hemoglobin, referred to hereinafteras Hb-AGE, is reviewed in further detail and particularly with regard toits capability as a diagnostic tool. Accordingly, Hb-AGE exhibits abroad range of activity, in that Hb-AGE and/or its antibodies may beused not only in detecting the onset of either glycemic conditions ordiabetes mellitus, but also in the long term monitoring of the course ofsuch conditions, to detect fluctuations in the intensity of suchconditions. Likewise, therapeutic applications for both Hb-AGE and itsantagonists are contemplated.

The present invention also extends to the measurement of urinary- andserum AGE-peptides and to the diagnostic and therapeutic applicationsthat follow. Urinary AGE-peptide levels are indicative of the turnoverof tissue AGEs and are therefore also useful in the evaluation ofdiabetes and diabetic complications, and particularly, the control ofblood glucose levels, both short term and long term, e.g., up to about60-90 days. Serum AGE-peptides are predictive of renal disease, andparticularly may be measured to determine glomerular filtration rate(GFR). Accordingly, diabetic complications involving the kidney can bemonitored and serum AGE-peptides can be used to evaluate the effect oftest compounds on the kidney and the level of AGEs elsewhere in thebody. Serum peptide-AGEs are also useful for detecting the AGE-inducingor -forming effect of drugs, and in evaluating the therapeutic effect ofAGE inhibitors, since serum peptides are derived from long-livedproteins.

Accordingly, the present invention relates to the measurement of Hb-AGE,serum AGE-peptides and urinary AGE-peptides, and to the associatedmethods for both the long-term and short-term monitoring of conditionsinvolving either sugar concentrations or AGE concentrations. Theinvention also includes the measurement of mammalian and particularlyhuman serum albumin AGEs (AGE-HSA), as well as food products for theassessment of spoilage, and proteins including recombinant preparations,that are intended for use as therapeutic agents. In this last mentionedconnection, the invention includes the use of the present AGE antibodiesin a method for monitoring the purity of such protein preparations, anda related method for the purification of the preparations that removeglycosylated proteins therefrom. Such a method would limit the unwantedadministration of glycosylated proteins that are known to be clinicallyactive in a manner deleterious to the host.

The method of assessing the presence or activity of disease in whichHb-AGE is the marker comprises obtaining a blood or other serum samplefrom a mammal, determining the presence or amount of Hb-AGE in thesample and comparing this amount to a standard.

Hb-AGE measurements provide an appropriate index of long-term tissuemodification by AGEs and are useful in assessing the contribution ofadvanced glycosylation to a variety of diabetic and age-relatedcomplications. While hemoglobin A_(1c) (HbA_(1c)) has been reported aspredictive of the extent of glycation on the hemoglobin β chain,HbA_(1c) is only an intermediate in the advanced glycosylation pathwayand numerous other intermediates are believed to exist. Moreover,HbA_(1c) is not predictive of pathology. Therefore, Hb-AGEs levels arebelieved to be a better measure of disease, drug effectiveness, etc.Hb-AGEs are used in the present invention to more readily correlate tothe progression of disease and longer term control of blood sugarlevels, which is greater than about 3-4 weeks. The reduction in Hb-AGElevels as a result of aminoguanidine therapy is a primary example of thedetection of successful pharmacological inhibition of advancedglycosylation in human subjects.

The invention also extends to the embodiment thereof that is common tothe present disclosure and that of parent application Ser. No.07/811,579, now abandoned, concerning antibodies which react with invivo-produced advanced glycosylation endproducts. Included therefore, isan antiserum that contains antibodies reactive with in vivo-formedadvanced glycosylation endproducts and has the followingcharacteristics:

A. it reacts with an immunological epitope common to in vivo-formedadvanced glycosylation endproducts;

B. it is cross reactive with advanced glycosylation endproducts formedin vitro; and

C. it is not cross reactive with the following advanced glycosylationendproducts however formed: FFI, AFGP, pyrraline, and pentosidine.Particularly, the common epitope is formed by the incubation of areducing sugar with a proteinaceous material selected from the groupconsisting of RNAse, lysine, hemoglobin, collagen Type IV, LDL, BSA andHSA.

The present application further reconsiders the reactivity of theantibodies of the invention with the AGE carboxymethyllysine. Althoughthe data presented herein indicate low, if any, reactivity of theantibodies with this AGE, subsequent evaluations with antibodies of theinvention suggest some recognition of this AGE. Accordingly, the presentapplication relates to detection of AGEs including carboxymethyllysine,a characteristic that is inherent to the antibodies of the invention asdisclosed herein.

The antibodies of the invention may be polyclonal or monoclonal, and ifthe latter, may be prepared by the hybridoma method, or other knownrecombinant techniques. As illustrated herein, the antibodies may beraised in an immunocompetent mammal by hyperimmunizing said mammal withAGEs or a protein on which AGEs have been formed. The antibodiesproduced recognize and bind to in vivo-formed AGEs, samples whichcontain such AGEs, e.g., diabetic tissue or serum, as well as invitro-formed AGEs which form on proteins as a result of incubation withsugars.

The anti-AGE antibodies of the invention are likewise characterized inthat they do not recognize certain AGEs that have been syntheticallyproduced, e.g., FFI, pyraline, AFGP, and pentosidine.

The anti-AGE antibodies described herein are further characterized asfollows:

(a) the antibodies can be formed by hyperimmunization of a mammal withAGE-RNAse;

(b) the antibodies are reactive with the following AGEs: AGE-RNAse,AGE-hemoglobin, AGE-BSA, AGE-HSA, AGE-collagen IV, AGE-LDL andAGE-lysine;

(c) the antibodies are non-reactive with unmodified HSA or BSA, FFI-BSA,formylated-albumin, maleylated-albumin, LDL, collagen IV, acetyl-LDL,FFI, AFGP, pyrraline, pentosidine, lysine, deoxypropylaminofructose ordeoxymorpholinofructose.

In a further aspect of the invention, the present anti-AGE antibodiesmay be recovered from an antiserum raised in a suitable host which hasbeen inoculated with a particular AGE-protein. The preferred methodcomprises administering to an immunocompetent mammal an effective amountof an AGE or a compound containing AGEs as above described, to inducethe formation of the present anti-AGE antibodies, and obtaining from themammal a serum which contains the anti-AGE antibodies. A particularAGE-protein comprises the product of the incubation of RNAse withglucose.

Another aspect of the present invention relates to immunological assaysfor detecting the presence or quantity of AGEs in a sample, comprisedof:

(a) binding a sample suspected of containing AGEs, an AGE carrier,anti-AGE antibodies or another AGE binding partner to a solid support;

(b) contacting the specie attached to the solid support with an analyteto be tested for the presence or quantity of AGEs, anti-AGE antibodiesor other AGE binding partner;

(c) labelling the AGEs, anti-AGE antibodies or other binding partnerwith a detectible label; and

(d) comparing the amount of bound label to a standard.

The above assay format may be adapted to examine samples of plant andanimal matter for the presence of AGEs, for example to detect thelikelihood or onset of food spoilage, and the present invention isintended to extend to this utility.

The invention further encompasses the use of the present antibodies forthe detection of disease in a mammal, which is characterized in that anabnormal level of AGEs such as Hb-AGE, serum AGE peptides and urinaryAGE peptides, is present. The antibodies may be either polyclonal ormonoclonal, and are as characterized earlier herein.

Assay kits are also encompassed which are useful for performing theassay/diagnosis described herein, which include suitable reagents fordetecting or quantifying AGEs such as Hb-AGE, urinary- and serumAGE-peptides, AGE-antibodies or other AGE binding partners in a sample.

Therapeutic compositions and methods of use in the prevention, diagnosisor treatment of disease using these compositions are also included,wherein an effective amount of the composition is administered to apatient in need of such treatment.

Consequently, a primary object of the present invention is to provide anantiserum which contains antibodies which recognize and bind to invivo-formed AGEs, and a method of making the antiserum containinganti-AGE antibodies which have not heretofore been produced.

Another object of the invention described herein is to provideimmunochemical assay protocols using the antiserum described above, forproteins which are modified by advanced glycosylation. AGE-immunogenshave been prepared in vitro without the use of chemical reduction andhydrolysis procedures, and antisera have been produced in vivo usingthese AGEs.

Another object of the present invention is to provide immunoassayprotocols which encompass the use of polyclonal antibodies raised inresponse to an immunogenic challenge with AGEs, as well as monoclonalantibodies all of which are specific to in vivo AGE epitopes.

It is a further object of the present invention to provide a method formeasuring advanced glycosylation endproducts in a variety of biologicalsamples that is rapid and reliable, taking advantage of the anti-AGEantibodies which have been raised and characterized.

It is a further object of the present invention to provide kitscontaining suitable reagents for the measurement of AGEs including asthe internal standard a material selected from Hb-AGE, serumAGE-peptides and urinary AGE-peptides, which are suited to a broad rangeof alternative immunological protocols.

It is a still further object of the present invention to provide amethod for the long term monitoring of glycemic conditions includingdiabetes, by the measurement of AGEs such as Hb-AGE, serum AGE-peptidesand urinary AGE-peptides, that is fast and reliable.

These and other objects will be apparent to the ordinarily skilledartisan from a review of the detailed description taken in conjunctionwith the following drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an antiserum dilution curve for anti-AGE-RNAse antiserum.Antiserum was titered in a noncompetitive ELISA utilizing the followingabsorbed antigens: RNAse (∘), Glucose-derived AGE-RNAse (), BSA (Δ),Glucose-derived AGE-BSA (▴), and G6P-derived AGE-BSA (▾);

FIGS. 2A-2C are ELISA competition curves for anti-AGE-RNAse antiserum.Assays were performed as described in Materials and Methods and employedglucose-derived AGE-BSA as the absorbed antigen. All points representthe mean of triplicate determinations.

FIG. 2A: Reaction with a variety of modified albumins. Glucose-derivedAGE-BSA (), G6P-derived AGE-BSA (▴), Fructose-derived AGE-BSA (▾),FFI-BSA (Δ), Formylated-BSA (∇), Maleyl-BSA (⋄), and BSA (∘).

FIG 2B: Reaction with AGE-modified and unmodified proteins. G6P-derivedAGE-HSA (), Glucose-derived AGE-LDL (▴), Glucose-derived AGE-collagenIV (♦), Glucose-derived AGE-RNAse (▪), HSA (∘), LDL (Δ), acetyl-LDL (∇),collagen IV (⋄), RNAse (□). LDL nmoles were calculated on the basis ofthe molecular weight of apoprotein B.

FIG. 2C: Reaction with model AGEs. Glucose-derived AGE-lysine (),G6P-derived AGE-lysine (♦), FFI-HA (∘), pyrraline (▴), AFGP (▾),carboxymethyllysine (▪), pentosidine (□), deoxypropylaminofructose (Δ),and deoxymorpholinofructose (∇). AGE-lysine products were added asnmoles of lysine equivalents;

FIG. 3 is the kinetic relationship between the formation ofAGE-associated fluorescence and antibody reactive material. BSA (50mg/ml) was incubated with glucose (0.5M) as described in Materials andMethods. Aliquots were sampled at indicate times, dialyzed to removeunbound material, and assayed for fluorescence (λ_(excitation) =370 nm,λ_(emission) =440 nm) and AGE content by ELISA;

FIG. 4 is the inhibition of the formation of antibody-reactive AGEs byaminoguanidine. BSA (100 mg/ml) incubated with glucose (100 mM) for 21days at 37° (∘). BSA (100 mg/ml) incubated with glucose (100 mM) in thepresence of 100 mM aminoguanidine for 21 days at 37° (). Bufferconditions were as described in Materials and Methods;

FIGS. 5A-5B are the determination of AGE-collagen in experimental rats.Diabetes was induced in Lewis rats with either alloxan or streptozotocinas described in Materials and Methods. At 16 week intervals, 6 animalswere sacrificed and the aortic collagen analyzed for hydroxyproline,fluorescence, and AGE-content by ELISA. Values are expressed per mg ofhydroxyproline.

FIG. 5A: Relative fluorescence measured at λ_(excitation) =370 nm andλ_(emission) =440 nm).

FIG. 5B: Collagen-bound AGEs measured by ELISA. Control rats (∘), ratswith alloxan-induced diabetes (▴), and rats with streptozotocin-induceddiabetes (▾). Each value shown is the mean of six experimental animals;

FIG. 6 is the determination of human serum AGE levels. NL: Normalindividuals (n=12). DM: Diabetic individuals (n=21). DM+HD: Diabeticindividuals on hemodialysis (n=16). Error bars show the S.E.M. P<0.001for DM vs. NL. P<0.001 for DM+HD vs. DM.;

FIG. 7 is a graph comparing Hb-AGE and HbA_(1c) levels in red bloodcells of normal and diabetic patients and thereby depicting thecorrelation between the two (normal patients=▪) (diabetic patients=□);

FIGS. 8A-8B comprise two bar graphs that show and compare the mean ofHb-AGE (FIG. 8A) and HbA_(1c) (FIG. 8B) levels in normal and diabeticpatients;

FIG. 9 is a graph depicting the results of the ELISA of Hb-AGE levelsmeasured in 23 diabetic individuals (DM) and 9 non-diabetic,normoglycemic individuals (NL). NL: 4.3±0.3 AGE Units/mg Hb, DM: 7.7±0.6AGE Units/mg Hb, (Mean±S.E.). Each value represents the mean oftriplicate determinations, assayed at 3-4 hemoglobin dilutions to ensurethat measured values fell within the linear range of the ELISA standardcurve. AGE units are calculated relative to an AGE-albumin standardsynthesized and analyzed for AGE content as described.

FIG. 10 graphically shows a correlation between levels of Hb-AGE andHbA_(1c) for nine normoglycemic (∘) and 23 diabetic () individuals.HbA_(1c) was measured by HPLC.

FIG. 11 is a graph of Hb-AGE formation in vitro. Human hemoglobin (50mg/ml) (Sigma Chemical Co.) was incubated at 37° C. in 0.4M NaPO₄ buffer(pH 7.4) containing 0 mM glucose (∘), 5 mM glucose (), 20 mM glucose(▴), or 20 mM glucose and 50 mM aminoguanidine (♦). Aliquots containinghemoglobin and 20 mM glucose were reduced with a 200-fold molar excessof sodium borohydride prior to ELISA (∇). Aminoguanidine (50 mM) alsoinhibited (>95%) Hb-AGE formation in the 5 mM glucose condition (datanot shown). Aminoguanidine (50 mM) did not inhibit the detection ofHb-AGE when added to glucose/hemoglobin incubations 1 hour prior toELISA analysis (data not shown). All incubations were performed aftersterile filtration. One ml samples were removed at the indicated timepoints and dialyzed against phosphate-buffered saline prior to ELISAanalysis.

FIG. 12 is a determination of Hb-AGE and HbA_(1c) levels in 18 diabeticpatients before (▪) and after (▪) 28 days of aminoguanidine therapy.Hb-AGE: 13.8±0.8 U AGE/mg Hb→10.0±0.9 U AGE/mg Hb; HbA_(1c) :10.1%±0.8%→9.2%±0.8% (Mean±S.E.).

DETAILED DESCRIPTION OF THE INVENTION

Numerous abbreviations are used herein to simplify the terminology used,and to facilitate a better understanding of the invention. The followingabbreviations are representative.

As used herein, the terms "AGE" and "AGEs" are used as appropriate torefer to advanced glycosylation endproducts which are in the form ofintermediates and stable compounds which are produced in vivo and invitro by the reaction of reducing sugars with protein amino groups. AGEstherefore encompass intermediates as well as stable endproducts that areimplicated in the structural and functional alteration of proteins seenduring aging. For example, AGEs are recognized to react with freepolypeptide amino groups, which leads to protein crosslinking.Additionally, such AGEs are observed in elevated levels in circulationand in tissues in certain diseases, e.g. diabetes mellitus.

When the designations "AGE-RNAse", "AGE-Hb", "AGE-BSA", "AGE-HSA","AGE-albumin", "AGE-collagen" and "AGE-LDL" are used, each refers to theadvanced glycosylation endproducts which are formed upon chemicalreaction of the substrates RNAse, Hb, BSA, HSA, albumin, collagen andLDL, respectively with one or more reducing sugars. Thus, AGE-RNAserefers to the advanced glycosylation endproducts of the reaction betweenbovine ribonuclease and a reducing sugar.

Albumin, when recited generically, refers to any specie from which itwas obtained, e.g., human, bovine, etc.

BSA refers to bovine serum albumin.

HSA refers to human serum albumin.

RNAse refers to ribonuclease generally, and where appropriate, to bovinepancreatic ribonuclease in particular.

Collagen is used in the conventional sense to refer to any type ofcollagen and derived from any appropriate source. When a specific typeof collagen was used, such as in the example, the particular type isnoted. However, it is recognized that alternative collagen types canalso be used.

LDL is also used in the conventional sense to refer to low densitylipoprotein.

FFI-BSA refers to a model AGE-protein produced by incubating2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole hexanoic acid with bovineserum albumin and coupling the reactive compounds withdicyclohexylcarbodiimide. Formaldehyde-BSA, maleyl-BSA and acetyl-LDLall refer to modified proteins produced as described below.

The present invention relates to the measurement of certain invivo-generated AGEs and particularly Hb-AGE, serum AGE peptides andurinary AGE peptides, and to the diagnostic and therapeutic applicationsto which such measurement may be put. The use of Hb-AGE as a markerfacilitates the long-term measurement of blood sugar levels and theconsequent ability to monitor glycemic conditions such as exist indiabetes mellitus. As the data presented herein reveals, Hb-AGE incontrast to the known determinant HbA_(1c), decreases in response to theadministration of the in vivo glycation inhibitor aminoguanidine andthus represents a clinically accurate time-integrated parameter ofrecent blood sugar levels that possesses the added dimension of use forboth diagnostic and therapeutic purposes.

Particularly, the present invention takes advantage of a competitiveELISA developed with the anti-AGE antibodies that form a part hereof andthat are also comprehensively presented in parent application Ser. No.07/811,579, and comprises a quick and effective diagnostic method. Asshown in FIG. 9, Hb-AGE represents a stable and reliable glucose-derivedrearranged Amadori product around which a diagnostic protocol may bedeveloped and carried out. Accordingly, the invention extends to amethod for the long term monitoring of in vivo glycemic conditions andthe concomitant assessment of the efficacy of any control measures thatmay be implemented.

The present invention also extends to the measurement of other AGEs andparticularly serum and urinary AGE-peptides. Serum and urinaryAGE-peptides, like Hb-AGE, represent circulating markers of AGEaccumulation that reflect the onset and extent of pathologies and otherdysfunctions where such accumulation is a characteristic. Thus, thoseage-related and diabetic conditions where increased levels of AGEs havebeen observed, such as, for example, atherosclerosis, cataracts anddiabetic nephropathy, may be monitored and assessed over the long termby the measurement of these AGEs, particularly by resort to thediagnostic methods disclosed herein.

Likewise, the methods including Hb-AGE or the noted AGE-peptides asmarkers or determinants may be used as a drug discovery assay, foridentification of drugs or other modalities that may interact with theseAGEs. In the instance of Hb-AGE, it may be possible to discover agentsthat may control either the concentration of blood glucose or theformation of Hb-AGE itself. An assay or test kit in such instance wouldinclude the reagents set forth for such kits presented later on herein,with Hb-AGE as the ligand or binding partner. A similar assay or kit maybe prepared with the noted AGE peptides serving in like capacity toHb-AGE.

The investigation of AGE formation in vivo has been hampered by a lackof specific assay methods and by the general inaccessibility of tissueAGEs to in vivo analysis. The realization that Amadori products such asHbA_(1c) are AGE precursors led to a consideration that hemoglobin mightalso acquire AGE modifications that could be measured with recentlydeveloped ELISA techniques.

Hb-AGE has been determined to account for about 0.42% of circulatinghuman hemoglobin. This fraction increases to approximately 0.75% inpatients with diabetes-induced hyperglycemia. Of significance, diabeticpatients treated for 28 days with aminoguanidine, an inhibitor of AGEformation in vivo, showed significantly decreased levels of Hb-AGE atthe end of the treatment period.

The tissue and end organ damage caused by advanced glycosylationaccumulates over a period of months to years. Diabetic complicationsprogress over a similar duration.

As stated above, a particularly preferred aspect of the presentinvention is the use of Hb-AGE as an indicator of the extent of controlof blood glucose in diabetes patients. Taking into account the normalHb-AGE levels and the reaction time over which Hb-AGE is formed, thelevel of Hb-AGE present in the blood can be predictive of diseases andunderlying pathology where the level of Hb-AGE is above normal values.

Similarly, serum peptide-AGEs can be used as an indicator that reflectsglomerular filtration rate (GFR) and kidney damage. Urinary AGE-peptidesmay be used as an indicator to measure the turnover in tissue proteins,and more particularly, tissue-AGE proteins.

In both the Hb-AGE and the serum AGE-peptide assays, the blood sample isdrawn and a separation procedure can be used. The cellular bloodcomponents can be separated from the serum, and in the Hb-AGE assay, thehemoglobin can be extracted from the red blood cells. The serum level ofAGE-peptides and the presence or extent of Hb-AGEs present can then beevaluated.

By conducting both tests with a single blood sample, a broader timeframe at which blood glucose levels become uncontrolled can beestimated, e.g., a 60 day range predictable by Hb-AGE that extends tothe periods before, during or after the 3-4 week time frame which ispredicted by Hb-A_(1c). If desired, the analyses of Hb-AGE and serumAGE-peptides can also be run together with a glucose level determinationin blood or urine, a glucose tolerance test, and other tests useful forassessing diabetes control including the measurement of urinaryAGE-peptides, to give a complete patient profile.

Another aspect of the invention addresses advanced glycosylationendproducts which can be detected in the urine. Proteins, includingpeptides, are excreted in the urine in low levels in normal individuals,and in perhaps elevated levels in diseased individuals. The presenceand/or level of urinary AGE-peptides reflective of the turnover oftissue AGEs can be determined, correlated to and predictive ofparticular diseases or conditions. For example, the quantity of peptidesfound in normal urine usually ranges from about 25 to 50 mg per day andis comprised of microproteins, with properties which are quite differentfrom the predominant blood proteins, albumin and globulin.

The presence of proteins in the urine may be a symptom of numerousdiseases or conditions reflective of a net catabolic state as wouldexist when the host or patient is undergoing invasion as by infection.Under such circumstances, the host mobilizes against the invasivestimulus by the secretion of numerous factors such as cytokines thatsuspend the anabolic energy storage activity and cellular repairactivities and promote instead the catabolic depletion of energy storesand the recruitment of leukocytes and other factors to fight andneutralize the stimulus. As this activity results in a correspondingelevation in tissue protein levels, and corresponding levels oftissue-AGEs, the measurement of urinary AGE-peptides provides yetanother index of possible invasive activity in the host, such ascachexia and shock. Thus, one can measure the presence or level ofAGE-peptides in urine, and correlate this level to a standard. In normalindividuals, the normal level may be at or close to zero. In diabeticpatients or in patients experiencing infection or other trauma, thenormal level of AGE-peptides may be significantly greater. Thus, theadvancement or worsening of diabetes prior to the onset of renalcomplications, or the presence of infection could be detected bydetecting increases in urine levels of AGE-peptide.

Likewise, one may be able to detect individuals who glycosylate proteinsfaster than normal. In this instance, one would determine the level ofAGE-peptides in the urine as a result of a specific challenge, e.g.,with glucose, a compound which induces AGE formation or release by theproteins of the body, or the rate at which urinary peptide-AGE levelsincrease after cessation of the administration of an AGE inhibitingcompound, e.g., aminoguanidine. A full clinical picture will thereforebecome apparent.

The present invention also relates to detecting the presence or level ofsuch AGE-peptides in serum. This may take into account the extent of AGEaccumulation and reaction with extracellular and cellular bloodproteins, protein fragments and peptides (shorter chain amino acids,e.g., up to about 50 amino acids in length) found in the circulatorysystem. These can be evaluated for the presences or level of AGEs, theextent of advanced glycation determined, and compared to a standard,e.g., normal peptide glycation levels. For example, if one detects anelevated level of AGE-peptides in the blood, one may correlate this tothe extent of kidney damage sustained by the patient.

Amadori products are slowly reversible and are believed to attainequilibrium over a 3-4 week period. AGEs, in contrast, remainirreversibly attached to proteins and continue to accumulate over thelifespan of the protein. The utility of Hb-AGE measurements as a longterm in vivo marker of advanced glycosylation can thereby beappreciated.

As stated earlier, the invention also comprises the identification of anantiserum and corresponding antibodies that recognize and bind to invivo-formed advanced glycosylation endproducts. As demonstrated in theexamples presented later on herein, polyclonal AGE-ribonucleaseantiserum has been prepared and used in competitive ELISA systems tostudy the specificity of this antiserum for in vitro- and invivo-derived products. The resulting antiserum recognized and bound toin vivo AGEs in diabetic tissue and serum known to contain abnormallyelevated levels of AGEs, leading to the conclusion that a common epitopeexists among them. By contrast, each of the model, synthetic AGEs whichwere synthesized and tested failed to react with the polyclonalantiserum, although other in vitro-formed AGEs are reactive.

Examples of proteins and protein-containing substances suitable forincubation-type reactions with the reducing sugars include, for example,RNAse, hemoglobin, collagen, BSA and HSA, each of which can be incubatedwith, e.g., glucose, glucose-6-phosphate ("G6P"), fructose or ribose toproduce a suitable AGE immunogen for inducing the formation of anti-AGEantibodies.

Such anti-AGE antibodies can also be used in the treatment of patientsto reduce the level of circulating AGEs or AGEs which may be present inabnormally elevated levels in certain tissues, e.g., pancreas, liver,kidney or brain.

Additionally, it is within the scope of the invention described hereinto utilize the anti-AGE antibodies for the design, screening and/orpotentiation of drugs or compounds which are useful for treatingelevated levels of AGEs in vivo. In this connection, the anti-AGEantibodies may be used to purify proteins that have been speciallycultivated or produced for use as therapeutic agents. As stated earlier,the therapeutic use of such proteins is increasing in prominence andimportance, and such proteins like other host cells, are susceptible toglycation and the formation of AGEs. As such AGEs are particularlyclinically active, it is desirable to limit their introduction into ahost during therapy. As a consequence, the present invention includes amethod for purification of batches of such proteins by bringing theminto contact with, for example, a quantity of anti-AGE antibodiesimmobilized on a suitable substrate. In this way the glycosylatedproteins could be separated from the rest of the batch by conventionalprocedures. The substrate could be refreshed or replaced periodically inthe instance of a commercial process, so that a continuous circulationof protein material past the substrate and subsequent separation of theprotein-AGE component could be conducted. Naturally, the foregoingscheme is presented for purposes of illustration only, and is capable ofvarious modifications in design and execution within the skill of theart and the scope of the invention.

The invention also includes methods for measuring protein aging both inplants and in animals, by assaying the presence, amount, location andeffect of such advanced glycosylation endproducts utilizing the anti-AGEantibodies. By reacting anti-AGE antibodies with samples of productssuspected of containing AGEs, plant matter and animal food samples canbe evaluated to assess food spoilage and the degradation of theproteinaceous materials so affected, while the assays of animals,including body fluids such as blood, plasma and urine, tissue samples,and biomolecules such as DNA, that are capable of undergoing advancedglycosylation, assist in the detection of pathology or other systemicdysfunction.

Further, such assay may be employed to assess the extent of degradationof proteins that have been cultivated, harvested or otherwiserecombinantly prepared for therapeutic use, to determine whether and/orto what extent such materials have become glycosylated. This assay couldbe used alone or in conjunction with a purification procedure, so thatthe determination that the protein material has developed apredetermined threshold level of glycosylation would signal the need forperforming a purification procedure such as that described above, on abatchwise or continuous basis.

The assay methods of the invention comprise the performance of severalassay protocols, involving the anti-AGE antibodies described hereinwhether labeled or not, the analyte, and/or a ligand, one or morebinding partners to the antibody, and binding partners thereto asapplicable.

The term "binding partners" is used in the general sense to refer tocomponents used in the assays which recognize and/or bind to each other.Thus, an anti-AGE antibody and the AGEs recognized by the antibody areconsidered binding partners.

The term "binding partners" also includes ligands useful in the presentinvention, such as AGE derivatives that bind to AGE binding partners.These ligands may be detected either singly and directly, or incombination with a second detecting partner such as avidin or biotin.Suitable synthesized AGE derivatives are selected from the reactionproducts of reducing sugars, such as glucose, glucose-6-phosphate (G6P),fructose and ribose and peptides, proteins and other biochemicals suchas BSA, avidin, biotin derivatives, and enzymes such as alkalinephosphatase.

Useful with this invention are enzymes and other carriers that haveundergone advanced glycosylation. These AGE-enzymes may serve as thepreferred labelled ligands in the assays of the present invention. Othersuitable ligands may include the reaction product of the reducing sugarsdirectly with carriers capable of undergoing advanced glycosylation.Suitable carriers may be comprised of a material selected fromcarbohydrates, proteins, synthetic polypeptides, lipids, bio-compatiblenatural and synthetic resins, and mixtures thereof.

The assays of the invention may follow formats wherein either the ligandor the binding partner are bound to a substrate. Likewise, the assaysinclude the use of labels which may be selected from radioactiveelements, enzymes and chemicals that fluoresce.

The present methods have particular therapeutic relevance in that meansfor the detection and evaluation of the condition of a broad spectrum oforgan systems are provided. The Maillard process acutely affects severalof the significant protein masses in the body, among them collagen,elastin, lens proteins, and the kidney glomerular basement membranes.These proteins deteriorate both with age (hence the application of theterm "protein aging") and as a result of prolonged exposure to elevatedblood sugar levels and AGE formation, the latter in turn frequently dueto pathology.

In this manner, the location and relative concentrations of advancedglycosylation endproducts in the body can be identified. Moreover, byassaying different organ specimens, an AGE "patient profile" can beobtained, i.e., the location and relative concentration of AGEs in thepatient can be identified. This technique is particularly useful inidentifying abnormal concentrations of advanced glycosylationendproducts, such as in atheromatous plaques. In such manner, thelocation of future systemic malfunctions can be identified.

Accordingly, the present assay method broadly comprises the steps of:

A. preparing at least one sample suspected of containing said advancedglycosylation endproducts;

B. preparing at least one anti-AGE antibody directed to said samples,wherein the anti-AGE antibody is reactive with in vivo-produced advancedglycosylation endproducts and has the following characteristics:

i. it reacts with an immunological epitope common to said in vivo-formedadvanced glycosylation endproducts;

ii. it is cross reactive with advanced glycosylation endproducts formedin vitro; and

iii. it is not cross reactive with the following advanced glycosylationendproducts however formed: FFI, AFGP, pyrraline, and pentosidine;

C. placing a detectible label on a material selected from the groupconsisting of said sample, a ligand to said anti-AGE antibody and saidanti-AGE antibody;

D. placing the labeled material from Step C in contact with a materialselected from the group consisting of the material from Step C that isnot labeled; and

E. evaluating the resulting sample of Step D for the extent of bindingof said labeled material.

Suitable analytes which can be evaluated may be selected from, e.g.,plant matter, edible animal matter, blood, plasma, urine, cerebrospinalfluid, lymphatic fluid, and tissue; and certain synthesized compounds,individually and bound to carrier proteins such as the protein albumin.The analyte may also comprise a synthetically derived advancedglycosylation endproduct which is prepared, for example, by the reactionof a protein or another macromolecule with a reducing sugar. Thisreaction product can be used alone or combined with a carrier.

The carrier may be selected from the group consisting of carbohydrates,proteins, synthetic polypeptides, nucleic acids, lipids, bio-compatiblenatural and synthetic resins, antigens and mixtures thereof.

The anti-AGE antibodies described herein can be used both to diagnosethe degradative effects of advanced glycosylation of proteins in plants,edible animal matter, and the like, and to detect the adverse effects ofthe buildup of advanced glycosylation endproducts. Such conditions asage- or diabetes-related hardening of the arteries, skin wrinkling,arterial blockage, and diabetic, retinal and renal damage in animals allresult from the excessive buildup or trapping that occurs as advancedglycosylation endproducts increase in quantity. Therefore, thediagnostic methods of the present invention seeks to avert pathologiescaused at least in part by the accumulation of advanced glycosylationendproducts in the body by monitoring the amount and location of suchAGEs.

Using the present invention, one can assess and/or detect the presenceof stimulated, spontaneous, or idiopathic pathological states inmammals, by measuring the corresponding presence of advancedglycosylation endproducts. More particularly, the presence or amount ofthe AGEs may be followed directly by assay techniques such as thosediscussed herein, through the use of an appropriately labeled quantityof the present anti-AGE antibodies or at least one of their bindingpartners, as set forth herein. Alternately, AGEs defining epitopesreactive with the present anti-AGE antibodies, could be synthesized andused to raise binding partners (or antagonists to such binding partners)that could in turn, be labeled and introduced into a medium to test forthe presence and amount of AGEs therein, and to thereby assess the stateof the host from which the medium was drawn.

Thus, both AGE receptors and any binding partners thereto that may beprepared, are capable of use in connection with various diagnostictechniques, including immunoassays, such as a radioimmunoassay, usingfor example, a receptor or other ligand to an AGE that may either beunlabeled or if labeled, then by either radioactive addition, reductionwith sodium borohydride, or radioiodination.

The general assay procedures and their application are all familiar tothose skilled in the art and are presented herein as illustrative andnot restrictive of the procedures that may be utilized within the scopeof the present invention.

The advanced glycosylation endproduct forms complexes with one or moreof the binding partners, and one member of the complex may be labeledwith a detectable label. The fact that a complex has formed and, ifdesired, the amount thereof, can be determined by applicable detectionmethods, e.g., IgG recognition and reaction with the complexes formed.

Suitable radioactive elements may be selected from the group consistingof ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵I, ¹³¹ I, and ¹⁸⁶ Re. In the instance where a radioactive label, such asprepared with one of the above isotopes is used, known currentlyavailable counting procedures may be utilized.

In the instance where the label is an enzyme, detection may beaccomplished by any of the presently utilized colorimetric,chemiluminescent, spectrophotometric, fluorospectro-photometric,thermometric, amperometric or gasometric techniques known in the art.The enzyme may be conjugated to the advanced glycosylation endproducts,their binding partners or carrier molecules by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Also, and in a particular embodiment of the present invention, theenzymes themselves may be modified into advanced glycosylationendproducts by reaction with sugars as set forth herein.

Many enzymes which can be used in these procedures are well known andcan be utilized. The preferred enzymes for detection are peroxidase,β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucoseoxidase plug peroxidase, hexokinase plus GPDase, RNAse, glucose oxidaseplus alkaline phosphatase, NAD oxidoreductase plus luciferase,phosphofructokinase plus phosphoenol pyruvate carboxylase, aspartateaminotransferase plus phosphoenol pyruvate decarboxylase, and alkalinephosphatase. U.S. Pat. Nos. 3,654,090; 3,850,752; and 4,016,043 arereferred to by way of example for their disclosure of alternativelabeling materials and methods.

A number of fluorescent materials are also known which can be utilizedas labels. These include, for example, fluorescein, rhodamine andauramine. A particularly preferred detecting material includes one ormore fluorescent labels on anti-rabbit antibodies prepared in goats andconjugated with fluorescein through an isothiocyanate.

One assay format contemplates a bound antibody to which are added theligand and the analyte. The resulting substrate is then washed afterwhich detection proceeds by the measurement of the amount of analytebound to the antibody. A second format employs a bound ligand to whichthe antibody and the analyte are added. Both of these formats are basedon a competitive reaction with the analyte, while a third formatcomprises a direct binding reaction between the bound analyte and theantibody. In the latter two formats, the extent of binding of theantibody is measured by a direct label or a labeled binding partner.

All of the protocols disclosed herein may be applied to the qualitativeand quantitative determination of advanced glycosylation endproducts andto the concomitant diagnosis and surveillance of pathologies in whichthe accretion of advanced glycosylation endproducts is implicated. Suchconditions as diabetes and the conditions associated with aging, such asatherosclerosis and skin wrinkling represent non-limiting examples, andaccordingly methods for diagnosing and monitoring these conditions areincluded within the scope of the present invention.

The present invention also includes assay and test kits for thequalitative and/or quantitative analysis of the extent of the presenceof advanced glycosylation endproducts. Such assay systems and test kitsmay comprise a labeled component prepared, e.g., by one of theradioactive and/or enzymatic techniques discussed herein, coupling alabel to an anti-AGE antibody or a binding partner as listed herein; andone or more additional immunochemical reagents, one of which may be afree or immobilized ligand, capable either of binding with the labeledcomponent, its binding partner, one of the components to be determinedor their binding partner(s). One of the components of the kits describedherein is typically an anti-AGE antibody in labeled or non-labeled form.

In a further embodiment of this invention, commercial test kits suitablefor use in one instance by food technologists, and in other instances bymedical specialists may be prepared to determine the presence or absenceof advanced glycosylation endproducts. As stated earlier, the kits maybe used to measure the presence of advanced glycosylation endproducts onrecombinant or other purified proteins, and particularly those destinedfor therapeutic use, to assay them in a first instance, and in a secondinstance, to assist in their further purification.

In accordance with the testing techniques discussed above, one class ofsuch kits will contain at least labeled AGE, or its binding partner asstated above, and directions, of course, depending upon the methodselected, e.g., "competitive", "sandwich", "DASP" and the like. The kitsmay also contain peripheral reagents such as buffers, stabilizers, etc.

Accordingly, a preferred test kit may be prepared for the demonstrationof the presence, quantity or activity of AGEs, comprising:

(a) a predetermined amount of at least one labeled immunochemicallyreactive component obtained by the direct or indirect attachment of theanti-AGE antibodies of the present invention or a specific bindingpartner thereto, to a detectable label;

(b) other reagents; and

(c) directions for use of said kit.

More specifically, the preferred diagnostic test kit may comprise:

(a) a known amount of a binding partner to an anti-AGE antibody asdescribed above, or a ligand thereof, generally bound to a solid phaseto form an immunosorbent, or in the alternative, bound to a suitabletag, or plural such end products, etc. (or their binding partners) oneof each;

(b) if necessary, other reagents; and

(c) directions for use of said test kit.

In a further variation, the preferred test kit may comprise:

(a) a labeled component which has been obtained by coupling the bindingpartner of the anti-AGE antibody to a detectable label;

(b) one or more additional immunochemical reagents of which at least onereagent is a ligand or an immobilized ligand, which ligand is selectedfrom the group consisting of:

(i) a ligand capable of binding with the labeled component (a);

(ii) a ligand capable of binding with a binding partner of the labeledcomponent (a);

(iii) a ligand capable of binding with at least one of the component(s)to be determined; and

(iv) a ligand capable of binding with at least one of the bindingpartners of at least one of the component(s) to be determined; and

(c) directions for the performance of a protocol for the detectionand/or determination of one or more components of an immunochemicalreaction between the anti-AGE antibody and a specific binding partnerthereto.

The present invention extends to the production of the anti-AGEantibodies in a mammal, and an antiserum containing said antibodies. Forexample and as illustrated herein, a mammal can be immunized with theincubation product of a reducing sugar and any protein which containsfree amino groups, and which is subject to Amadori rearrangement, thusyielding AGEs.

As used herein, the terms "immunized" and "hyperimmunized" refer to thespecific immunization protocol also described in detail later on herein,which is used to elicit the antibody response that yields the antiserumand antibodies of the present invention. The protein or the reactionproduct of the protein described above incubated with one or morereducing sugars can be used. Typically about four primary doses of theimmunogen are administered to an immunocompetent mammal in an amounteffective for inducing the formation of anti-AGE antibodies. Boosterimmunization doses may also be administered as appropriate.

The immunization of the host mammal with the protein itself or with AGEsderived from the AGE-protein, e.g., AGE-RNAse, produces antibodies whichare reactive to AGEs derived from the immunogen itself or AGEs which arederived from other proteins. For example, the administration ofAGE-RNAse produces polyclonal anti-AGE antibodies which react withAGE-RNAse, AGE-hemoglobin, AGE-BSA, AGE-HSA, AGE-LDL and AGE-collagenIV, but not with unmodified RNAse, BSA or HSA.

The antibodies raised as described above were also generally unreactivewith model AGEs produced synthetically through procedures involvingchemical hydrolysis or reduction, such as the model AGEs FFI, AFGP,pyrraline, and pentosidine. None of these model compounds was recognizedby the anti-AGE antibodies raised.

The invention described herein takes advantage of the epitope which ispresent on these in vivo-derived AGEs and on in vitro-generated AGEsproduced without specific chemical hydrolysis or reductive conditions.The epitope can be exploited in numerous processes for detecting AGEs onin vivo-derived as well as in vitro-derived material. For example, thebinding affinity of anti-AGE antibodies can be used in non-competitiveand competitive ELISA assays, as well as in other protocols whichutilize different immunoassay configurations.

Ribonuclease modified by long-term incubation with glucose was found tobe a suitable immunogen for the production of high-titre, anti-AGEantibodies against a variety of AGE-modified proteins. Advancedglycosylation endproducts prepared with glucose showed the greatestinhibition, followed by AGEs prepared with G6P and fructose. Both G6Pand fructose react with proteins at a faster rate than glucose toproduce brown, fluorescent AGEs.

Different glycosylating sugars such as glucose, G6P, and fructoseproduce antigenically cross-reactive epitopes when incubated withprotein in vitro. In contrast, the purification of particular productsfrom in vitro incubation mixtures of polypeptides or amines with glucose(FFI, AFGP, pyrraline) or from tissue glycosylated in vivo (pentosidine)resulted in compounds with no demonstrable cross-reactivity withanti-(AGE-RNAse) antiserum. This suggests that the model AGEs which havebeen described thus far are either antigenically minor products, or thatthe purification procedures which are typically used for isolating AGEsfrom a sample resulted in structural alterations, such that reactivitywith anti-AGE antibodies is essentially eliminated.

In vitro time course studies revealed that the characteristicfluorescence of advanced glycosylation precedes the development of theanti-AGE reactive moieties. Thus, the anti-AGE antiserum appears to bemost reactive with "late" advanced glycosylation endproduct(s) whichform after fluorophore formation.

The ligands useful in the present invention are preferably invivo-generated AGEs that bind to AGE binding partners. These ligands maybe detected alone or in combination with a second detecting partner suchas avidin and/or biotin. Suitable ligands when these signal amplifiersare used can be selected from the reaction products of reducing sugarssuch as glucose, G6P, fructose, ribose and the like with peptides,proteins and other biochemicals such as BSA, avidin, biotin, and enzymessuch as alkaline phosphatase.

As discussed earlier herein, the invention extends to monoclonalanti-AGE antibodies which are capable of preparation by hybridomatechniques, utilizing, for example, fused mouse spleen lymphocytes andmyeloma cells. Immortal, antibody-producing cell lines can also becreated by techniques other than fusion, such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus.

Specific polyclonal antibodies can be raised in different preferred hostspecies. Naturally, these antibodies are merely illustrative of antibodypreparations that may be made in accordance with the present invention.

Specific protocols are illustrated below as necessary. The protocolsdisclosed herein may be applied to the qualitative and quantitativedetermination of AGEs and to the concomitant diagnosis and surveillanceof pathologies in which the accretion of AGEs is implicated. Conditionssuch as diabetes and those associated with aging, such asatherosclerosis and skin wrinkling, represent non-limiting examples.Accordingly, methods for diagnosing and monitoring these conditions areincluded within the scope of the present invention.

The biochemical and biological reagents which are recited below areavailable commercially or prepared in accordance with recognizedprotocols. All publications cited herein are hereby incorporated byreference.

MATERIALS AND METHODS REAGENTS

The reagents used in the assays described below were obtained orprepared as follows:

Bovine pancreatic ribonuclease ("RNAse"), bovine serum albumin ("BSA"),human serum albumin ("HSA"), collagen Type 4, collagenase, glucose,glucose-6-phosphate ("G6P"), fructose, ribose, and sodium borohydridewere obtained from Sigma Chemical Corp. (St. Louis, Mo.).

AGE-hemoglobin was prepared by isolating red blood cells, hemolyzingthem with toluene and treating a sample of the red cell hemolysate withtrichloroacetic acid (TCA). Specifically, a 50-100 μl sample of RBChemolysate was prepared and 3 ml of water and 1 ml of 24% (wt/vol) TCAwere added. The mixture was agitated, and thereafter centrifuged for 30minutes at 3000 rpm. The resulting supernatant was aspirated, 150 μl 1NNaOH was then added, after which water was added to a total volume of0.5 ml. This material was then diluted from 1:2 to 1:200 with 0.3M KH₂PO₄ pH 7.4, and was prepared for performance of the assay.

AGE-albumin was prepared by incubating albumin (50 mg/ml) with 0.5Mglucose, G6P or fructose in 0.2M NaPO₄ buffer (pH 7.4) for 60 days.

AGE-collagen was synthesized by incubating collagen (5 mg/ml) with 0.5Mglucose in 0.2M NAPO₄ buffer (pH 7.4) for 21 days.

AGE-RNAse was prepared by incubating RNAse (25 mg/ml) with 1M glucose in0.2M NAPO₄ buffer (pH 7.4) for 90 days.

FFI-BSA was prepared by coupling FFI-hexanoic acid to BSA withcarbodiimide.

Formaldehyde-BSA, maleyl-BSA and acetyl-LDL were synthesized inaccordance with the procedures described in Horiuchi, S. et al., J.BIOL. CHEM., 4:260, 432, 438 (1985); Takata, K. et al., BIOCHIM.BIOPHYS. ACTA., 94:273-280 (1989); and Goldstein, J. L. et al., PROC.NATL. ACAD. SCI. USA, 77:333-337 (1979).

AGE-LDL was prepared according to a protocol in which spontaneousoxidation is minimized, according to Kirstein, M. et al, PROC. NATL.ACAD. SCI. USA, 87:9010-9014 (1990).

AGE-BSA was reduced with sodium borohydride as described in Fluckiger,R. et al, METHODS ENZYMOL., 106:77-87 (1984). Any unreacted borohydridewas removed by dialysis against PBS.

For aminoguanidine inhibition, BSA (100 mg/ml) was incubated with 100 mMglucose and 100 mM aminoguanidine hydrochloride (Aldrich Chemical Co.,Milwaukee, Wis.) in 0.2M NaPO₄ buffer (pH 7.4) for 21 days at 37° C.Samples were dialyzed against PBS prior to analysis.

Lysine-derived AGEs were prepared by incubating 1M glucose-6-phosphateor 1M glucose with 50 mM L-lysine in 0.2M sodium phosphate buffer (pH7.4) 10 days at 37° C.

1-deoxy-1-morpholino-D-fructose was obtained from Sigma Chemicals, Inc.and 1-deoxy-1-propylamino-D-fructose was prepared from an α-D-glucoseand N-propylamine according to Mitchel, F. et al., CHEM. BER.,92:2836-2840 (1959). 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole) wassynthesized from an aqueous mixture of furylglyoxal and 6-aminohexanoicacid and purified by medium pressure chromatography on silica gel.

1-alkyl-2-formyl-3,4-diglycosyl-pyrrole (AFGP) was prepared byincubating glucose with 6-aminohexanoic acid and sodium sulfite for 26days at 37° C. followed by chromatography on Dowex AG 1×4 anion exchangeresin and HPLC.

OTHER METHODS

The anti-AGE antibody and its reactivity with in vivo-derived AGEs, andits non-reactivity with AGEs synthesized chemically by methods involvinghydrolysis or reduction were evaluated. Protein concentrations weredetermined in accordance with Bradford, M., ANAL. BIOCHEM., 72:248-252(1976), and BSA was utilized as a standard. Ribonuclease and AGE-RNAseprotein amounts were determined additionally by SDS-PAGE and comparisonof Coomassie Blue stained bands with RNAse standards. Hydroxyprolinecontent was determined according to Edward, C. et al., CLIN. CHIM. ACTA,104:161-167 (1980). Collagen AGE-specific fluorescence determinationswere performed by measuring emissions at 440 nm upon excitation at 370nm using an LS-3B fluorescence spectrometer (Perkin-Elmer, Norwalk,Conn.).

A preparation of control albumin was also incubated under the sameconditions described above without sugar. All incubations were performedunder sterile conditions in the dark, and at 37° C. After incubation,unbound material was removed by extensive dialysis against phosphatebuffered saline (PBS) or by gel filtration over Sephadex G-10(Pharmacia, Uppsala, Sweden).

A single standard AGE-BSA preparation (1 mM AGE-BSA=12 A₃₅₀) was used asa reference. Fluorescence intensity standards were used to calibrate andmonitor the performance of the instrument. Fluorescence values of testsubstances were measured at a protein concentration of 1 mg/ml andexpressed as percent relative fluorescence compared to the AGE-BSAstandard.

PRODUCTION OF ANTI-AGE ANTIBODIES

Previous efforts to raise antisera against native AGEs generally havebeen unsuccessful or have failed to detect AGEs which occur in vivo.

To induce the formation of anti-AGE antibodies, synthesized AGEs wereproduced in vitro as described above without resort to hydrolyric orchemical reduction reactions. The protein was typically incubated with areducing sugar to form AGEs.

Glucose was preferably used as the glycosylating agent in vitro becauseit is the major circulating sugar and it produces AGEs in vitro whichclosely resemble the AGEs which are formed in vivo. RNAse was selectedas the target protein for advanced glycosylation because RNAse readilyforms AGEs and AGE-mediated intermolecular crosslinks.

Two female New Zealand White rabbits received four primary immunizationsand one booster immunization of RNAse or AGE-RNAse emulsified inFreund's complete adjuvant following a protocol for post-translationallymodified proteins in accordance with R. Bucala, et al., Mol. Immunol.,20:1289-1292 (1983) ("hyperimmunization protocol") and as follows.Accordingly, each rabbit received four intradermal injections over theback (200 μg each) and one injection in each hind quarter (100 μg each).This procedure was repeated at weekly intervals for six weeks. After atwo week rest, the rabbits received a booster injection of 1 mg ofantigen in Freund's incomplete adjuvant. The amimals were bled on thetenth day after this injection. Antibody response was monitored weeklyby Ouchterlony double diffusion and by non-competitive ELISA.

Using the hyperimmunization protocol described above, high titrepolyclonal rabbit anti-serum against RNAse (the carrier protein) wasobtained. The anti-RNAse titre was determined to be greater than 10⁻⁵ ina non-competitive ELISA. The following absorbed antigens were tested:RNAse, glucose-derived AGE-RNAse, BSA, glucose-derived AGE-BSA andG6P-derived AGE-BSA. The results are shown below in FIG. 1.

Significantly higher reactivity was observed for the immunogen AGE-RNAsethan RNAse. The anti AGE-RNAse antiserum also reacted with albuminmodified by incubation with either glucose or G6P, but not withunmodified albumin, indicating the presence of antibodies specific forAGEs.

EXAMPLE 1 ELISA ASSAYS

To monitor the formation of anti-AGE antibodies, rabbit antiserumproduced as described above, was titered in a non-competitive ELISAsystem. RNAse, AGE-RNAse, and AGE-BSA were used as the absorbedantigens.

The absorbed antigen was contacted with rabbit serum, allowing theanti-AGE antibodies contained in the serum to complex. The level ofcomplex formed was then evaluated by adding anti-rabbit IgG antibodiesconjugated to alkaline phosphatase (Organon Technica, Durham, N.C.). Thetitre for anti-(AGE-RNAse) antiserum was thereafter defined as the serumdilution giving a 50% of maximum OD₄₀₅ signal.

Ligand inhibition and AGE measurements were then performed incompetitive ELISA. Ninety-six well microtitre plates (Nunc Immunoplate,Gibco, Grand Island, N.Y.) were coated with AGE-BSA (obtained asdescribed above) by adding 0.1 ml of a solution of AGE-BSA (10 μg/ml,dissolved in PBS) to each well and incubating for 2 hrs. at roomtemperture. Wells were then washed three times with 0.15 ml of asolution containing PBS, 0.05% Tween-20, and 1 mM NaN₃ (PBS-Tween).

The wells were blocked by incubation for 1 hour with 0.1 ml of asolution of PBS containing 2% goat serum, 0.1% BSA, and 1 mM NaN₃. Afterwashing the blocked wells with PBS-Tween, 50 μl of a competing antigenwas added, followed by 50 μl of the rabbit-derived antiserum (finaldilution, 1/1000).

Plates were thereafter incubated for 3 hours at room temperature, afterwhich the wells were washed with PBS-Tween and developed with analkaline phosphatase linked anti-rabbit IgG (raised in goats) utilizingp-nitrophenylphosphate as the colorimetric substrate.

Results were expressed as B/Bo, wherein Bo is the maximum amount ofantibody bound in the absence of competing antigen, and B is the amountof antibody bound in the presence of competing antigen. Both Bo and Bhave been adjusted for background (See Robard, CLIN. CHEM., 20:1255-1270 (1974) and calculated as experimental optical density at 405nm-background optical density (no antibody)!/ total (nocompetitor)-background optical density!.

It was determined that three micrograms of the glucose-derived AGE-BSAstandard inhibited antiserum binding by 50%. This standard yielded anA₃₅₀ of 12 mM⁻¹ albumin.

The specificity of the anti-(AGE-RNAse) antiserum was thus tested usinga variety of AGE-modified proteins, unmodified proteins, and syntheticAGEs. As shown in FIG. 2(A) results were obtained by testinganti-(AGE-RNAse) antiserum against different modified albumins in acompetitive ELISA system using AGE-BSA as the absorbed antigen. Allpoints represent the mean of triplicate determinations.

The AGE-albumin prepared by incubation of BSA with glucose showed thegreatest inhibition, followed by AGE-albumin prepared withglucose-6-phosphate and with fructose.

FFI-BSA, a BSA derivative which carries the synthetic AGE ligand FFI,was not recognized by the antiserum. Other albumin modifications such asformylation or maleylation, which produce specific recognition signalsfor albumin uptake in vivo likewise did not demonstrate anycross-reactivity with the antiserum.

The AGE-modification competed for antibody binding in the competitiveELISA when it was present on diverse carrier proteins (FIG. 2, B). Thus,G6P-derived AGE-HSA, glucose-derived AGE-LDL, and glucose-derivedAGE-collagen (type IV), all demonstrated specific inhibition of antibodybinding to glucose-derived AGE-BSA. In contrast, unmodified HSA,unmodified LDL, and unmodified collagen did not so compete. Acetyl-LDL,a modified form of LDL which is specifically recognized and taken up bycellular scavenger receptors, also was not recognized by theanti-(AGE-RNAse).

The anti-AGE antiserum was also tested for competition against model,structurally defined AGEs (FIG. 2, C). The model AGE products testedwere FFI, AFGP, pyrraline, carboxymethyllysine, and pentosidine. Thesecompounds were isolated from in vitro incubations of amines withreducing sugars or from tissue collagen after reduction and acidhydrolysis. These compounds were tested at high concentrations so thateven low levels of inhibition would be readily detected. None of theseproducts competed with the binding of the antiserum to AGE-BSA, with theexception that at very high concentrations, carboxymethyllysinedemonstrated some inhibitory activity. This activity, whiledemonstrating a trend toward inhibition, was not highly significant.Nevertheless, the data could not conclusively rule out some reactivitywith carboxymethyllysine.

Glucose and G6P were incubated with lysine to determine whether lowmolecular weight AGEs could react with the antiserum obtained. Theseproducts were generated by the incubation of glucose or G6P with lysinein vitro without chemical reduction or hydrolysis. The resulting productwas tested for reactivity in the competitive ELISA.

Control incubations consisting of either sugar or lysine alone failed toshow any competition (data not shown). Two model Amadori products,deoxypropylaminofructose and deoxymorpholinofructose were also tested,and each compound failed to inhibit antiserum binding. Further evidencethat Amadori products are not recognized by the antiserum was providedby the fact that sodium borohydride reduction of AGE-BSA did notdiminish reactivity in the competition ELISA (data not shown).

To further characterize the nature of the in vivo-generated AGEs whichreact with the anti-(AGE-RNAse) antiserum, AGEs were synthesized in thepresence of the advanced glycosylation inhibitor aminoguanidine.Aminoguanidine is a hydrazine-like compound which reacts at anintermediate stage of the advanced glycosylation process, inhibiting theformation of protein-bound fluorescent products and crosslinks. As shownin FIG. 4, the inclusion of aminoguanidine in an in vitro incubation ofglucose and BSA significantly inhibited the formation of AGEs whichreact and bind to the anti-AGE antiserum.

EXAMPLE 2 AGE FORMATION KINETICS

The kinetic relationship between the formation of AGE-associatedfluorescence and the formation of products which bind to anti-AGEantiserum was also evaluated. FIG. 3 shows a time course for thedevelopment of fluorescence and anti-AGE antibody reactive material. BSAwas incubated with glucose, aliquots were removed at various intervals,dialyzed to remove unbound products, and then assayed. AGE-fluorophoreswere observed to form rapidly between 0 and 40 days and to precede theformation of antibody reactive products.

When measured by ELISA, AGEs were not detected until day 20, and thenformed rapidly between days 30 and 70. The formation of both AGEfluorophores and antibody reactive products eventually plateaued.

EXAMPLE 3 DIABETES EVALUATION

As stated above, the present invention affords a particularly effectivemeans for the detection and evaluation of diabetes as well as otherdisease states in which AGE levels are abnormal. Effective assessment ofthe presence and/or quantity of AGEs in diabetic tissue, and the use ofthe AGE assays described herein to characterize the overall condition ofa mammal known to be diabetic, are described below.

To determine whether tissue AGEs could be measured by anti-AGE ELISA,rats with experimentally-induced diabetes mellitus were evaluated.

Diabetes was induced in 8-week-old male Lewis rats by the rapidintravenous injection of alloxan (40 mg/kg) or streptozotocin (65mg/kg). Hyperglycemia was confirmed by assaying blood glucose. Bloodglucose was determined at 16 week intervals and averaged 20.5±2.4 mM inthe alloxan-treated animals (n=24) and 23.5±3.9 in thestreptozotocin-treated animals (n=24). There was no significant changein blood sugar levels with time in the control, alloxan-treated, orstreptozotocin-treated animals.

At 4 month intervals, 6 animals were sacrificed and the aortas removedand frozen at -80° C. for later analysis. Arterial tissue was slowlythawed, rinsed with PBS, and finely minced with scissors. Lipids wereextracted with acetone/chloroform (1:1) by shaking gently overnight at4° C. Samples then were dried by vacuum centrifugation and resuspendedin 0.2M NaPO₄ buffer (pH 7.4). Collagenase (Type VII) was then added ata 1/100(w/w) ratio and the mixture incubated for 48 hr at 37° C. withmild shaking. One drop of toluene was included to maintain sterility.Digested samples then were centrifuged at 15,000×g and the clearsupernatants used for fluorescence, AGE, and hydroxyprolinemeasurements.

In the diabetic animals (alloxan-plus-streptozotocin group), relativefluorescence increased from 13.7%±2.4% at 16 weeks to 23.7%±3.0% at 64weeks (P<0.001).

Fluorescence in the control, non-diabetic animals increased slightlyduring this time period (8.3%±1.0% to 9%±0.8%, not statisticallysignificant). Tissue and serum AGE values were expressed as AGE Units.One AGE Unit was defined as the amount of antibody-reactive materialthat was equivalent to that in 1 μg of the AGE-BSA standard. The Pvalues were calculated by the unpaired Student's t-test for comparisonbetween groups.

Analysis of the AGE content by ELISA showed an approximately two-foldincrease with time in the diabetic animals (4.8±0.5 U/mg at 16 weeksversus 10.5±1.8 U/mg at 64 weeks, P<0.001). Arterial AGE content alsoincreased with time in the control, non-diabetic rats although at a muchlower rate than in the diabetic animals (2.5±0.6 U/mg at 16 weeks versus4.3±0.4 U/mg at 64 weeks P<0.001).

Human serum was also obtained from normal and diabetic patients.Patients with compromised renal function were also studied because thisgroup of patients has been found to have markedly elevated levels ofcirculating, serum AGEs.

These circulating, serum AGEs are primarily in the form of low-molecularpeptides which are inefficiently cleared by hemodialysis therapy.

Serum samples were obtained from non-diabetic (n=12), diabetic (n=21),and diabetic patients on hemodialysis (n=16). Serum was dilutedthree-fold with PBS and filter sterilized through a 0.22 μm Milliporefilter, (Millipore, Bedford, Mass.) prior to analysis.

When expressed as AGE U/ml, the normal patients (non-diabetic, normalrenal function) had a mean level of 10.5±1.3 U/ml serum. The AGE levelswere elevated more than two-fold in the diabetic patients (24.7±2.4U/ml, P<0.001 for diabetic (DM) vs. normal (NL) and almost eight-fold indiabetic patients on hemodialysis (79.4±9.9 U/ml, P<0.001 for DM+HD vs.DM). These results correlate well with the findings of a recent studywhich utilized a radioreceptor assay to measure the AGE-peptide contentof serum obtained from diabetic patients and from diabetic patients onhemodialysis.

EXAMPLE 4

An assay of time-integrated blood glucose levels was performed using theprotocol followed in Example 3 above, with samples taken from normal anddiabetic patients. The results of the assay were compared against valuesthat are received when time-integrated blood glucose is measured usingthe known standard of HbA_(1c), and are presented in FIGS. 7 and 8.

Referring to FIGS. 7 and 8, it can be seen that the performance of thepresent assay with AGE-hemoglobin as the standard compares favorablywith the known determinant and standard HbA_(1c), and can be used in anyinstance where the latter test may be called for. The data measurementsthat were received are virtually identical and the clinical integrity ofthe present assay is consequently high.

A further diagnostic application of the present invention is in themeasurement of fructose-derived AGEs. The measurement offructose-derived AGEs is being recognized as a significant determinantof the rate of AGE formation, and the concomitant development and extentof the pathologies and other sequelae that have been associated withthis reaction, such as diabetes mellitis. Suarez et al., (1989) J. BIOL.CHEM., 264:3674-3679, and McPherson et al., (1988) BIOCHEMISTRY,27:1901-1907, both suggest that the presence and participation offructose in protein crosslinking fortells a significant role forfructose-derived AGEs that commends its measurement and control, andsignificantly, Ahmed et al., (1992) CLIN. CHEM., 38(7):1301-1303, statethat fructosylated Hb is incapable of effective diagnosis by presentlyknown clinical assays.

Accordingly, the present invention is appropriately extended to themeasurement of fructose-derived AGEs in a comprehensive effort to betterunderstand and treat the adverse effects of the reaction of theaccumulation of fructose-derived AGEs with body proteins.

EXAMPLE 5

Anti-AGE antibodies developed for the detection of in vivo-formed AGEswere used in a competitive ELISA to measure hemoglobin-linked AGEs inred cell hemolysates. FIG. 9 shows the results of this analysis for 32red cell samples obtained from diabetic individuals (DM) andnon-diabetic normoglycemic individuals (NL). Hemoglobin-linked AGEs weredetected in both groups of individuals, but significantly higher amountswere present in the diabetic group (NL n=9!: 4.3±0.3 Units AGE/mg Hb; DMn=23!: 7.7±0.6 Units AGE/mg Hb, Mean±S.E.!, P<0.001 by Student'sunpaired t-test).

Antibody reactivity, expressed in AGE Units, was calculated relative toa synthesized AGE-albumin standard. Additional experiments showed thatthe hemoglobin-AGE modification is stable to dialysis, acidprecipitation and proteolysis, and is unaffected by borohydridereduction (data not shown). These data, together with previous studiesof anti-AGE antibody specificity confirm that the hemoglobin-AGE moietyis a stable glucose-derived post-Amadori product. The levels of red cellHbA_(1c) correlate with the levels of Hb-AGE in a statisticallysignificant manner.

EXAMPLE 6

The formation of Hb-AGE from hemoglobin and glucose was confirmed invitro. Purified human hemoglobin was incubated at 37° C. with glucoseconcentrations that mimicked normoglycemia (5 mM) and hyperglycemia (20mM). Hemoglobin-AGE formed in a time and concentration dependent manner.See FIG. 11. That early Amadori glycation products are unreactive withanti-AGE antibodies was confirmed in the present system by theobservation that sodium borohydride reduction which alters the Amadoriproduct epitope did not affect the detection of Hb-AGE products onceformed. The addition of aminoguanidine prevented the formation ofhemoglobin-associated AGEs.

Hemoglobin-AGE measurements were performed in blood specimens obtainedfrom patients undergoing treatment with aminoguanidine. The patientgroup consisted of 18 individuals with long standing diabetes mellitus.Blood samples were obtained before and after 28 days of aminoguanidinetreatment and the Hb-AGE levels were determined by ELISA. As shown inFIG. 12, the mean Hb-AGE value decreased significantly as a result ofaminoguanidine therapy. (13.8±0.8 Units AGE/mg Hb vs. 10.0±0.9 UnitsAGE/mg Hb Mean±S.E.! P<0.001, by Student's paired t-test).

HbA_(1c) values were not affected by aminoguanidine treatment(10.1%±0.8% v. 9.2%±0.8%, Mean±S.E.! P=NS). No significant changes ineither the Hb-AGE or HbA_(1c) levels were observed in blood samplesobtained from a group of six patients receiving a placebo control (datanot shown).

The existence of an AGE-modified hemoglobin is noteworthy in severalrespects. First, AGEs generally have been considered to require a timecourse of months to years to form, even under hyperglycemic conditions.The present findings indicate that within the lifespan of circulatingred cells, e.g. about 120 days, significant amounts of AGE-modifiedhemoglobin are formed. If Hb-AGE Units, expressed relative to asynthetic AGE-albumin standard, are recalculated as a fraction of totalred cell hemoglobin, Hb-AGE appears to account for 0.42±0.07% ofcirculating hemoglobin. This level increases to a mean of 0.75±0.08% inthe diabetic group that was studied. These values contrast withcorresponding HbA_(1c) fractions of 5.8% and 8.9% for the normoglycemicand diabetic groups respectively.

The high amount of AGE accumulation on hemoglobin compared to connectivetissue or basement membrane collagen may reflect the receptor-mediatedturnover of connective tissue AGEs during normal remodeling or indicatean inherently enhanced rate of AGE formation on hemoglobin as a proteinsubstrate. Alternatively, circulating red blood cell hemoglobin may besusceptible to modification by reactive plasma AGEs which occur inelevated amounts in patients with diabetes, renal insufficiency or otherdisease conditions. Irrespective of the mechanism of formation, theapplication of quantitative ELISA methods to measurements of invivo-generated AGEs indicates that AGE formation occurs more rapidlywith hemoglobin than with connective tissue collagen.

Hb-AGE may thus serve as a useful biochemical index of advancedglycosylation in vivo. The formation of Hb-AGE reflects a time integralof blood glucose concentration that is significantly longer than thatestablished for HbA_(1c) (3-4 weeks). Four week pharmacologicalintervention with aminoguanidine was sufficient to lower significantly(28%) the Hb-AGE levels in a treated diabetic population.

Hb-AGE measurements may facilitate a variety of investigations into thepathophysiology of both diabetes and age-related complications. Thesewould include clinical studies aimed at elucidating the benefit ofstrict glucose control in preventing diabetic complications, as well asexperimental investigations of the role of advanced glycosylation in thepathogenesis of such diabetes- and age-related conditions asatherosclerosis, hypertension and renal disease.

EXAMPLE 7 Effect of Aminoguanidine on Urine AGE Levels in Normal andDiabetic Rats

Groups of normal and streptozotocin-diabetic rats were left untreatedfor 11 weeks. Half of the animals in each of the two groups were thenstarted on daily treatment with 70 mg/kg aminoguanidine hydrochloride(AG HCl), by gavage, and the other half of the animals with distilledwater by gavage. After 10 additional weeks, urines were collected fromall animals over a 24 hour period.

Urine samples were centrifuged and the supernatants were dilutedeight-fold with 0.3M potassium phosphate buffer, pH 7.4. Samples of thediluted urines were run in the AGE ELISA assay described above. Theresults of these measurements are set forth in the Table, below.

                  TABLE                                                           ______________________________________                                                       Urine AGEs: Units                                              Treatment      excreted per 24 hrs.                                           ______________________________________                                        Normal rats    5400 ± 1200                                                 Normal rats treated                                                                          2200 ± 190                                                  with                                                                          70 mg/kg/day                                                                  AG HCl                                                                        Diabetic rats  9400 ± 340                                                  Diabetic rats treated                                                                        4100 ± 1200                                                 with 70 mg/kg/day                                                             AG HCl                                                                        ______________________________________                                    

From the above, it was observed that twenty-one weeks of diabetesproduced a 1.7-fold increase in urinary AGE excretion over that ofnormal animals which was normalized by aminoguanidine administration.Aminoguanidine-treated normal rats showed a 60% inhibition of urinaryAGE excretion. The above data accordingly confirms the relevance andvalue of the measurement of urinary AGEs and AGE-peptides to monitorconditions such as diabetes where the turnover of tissue AGEs is aclinically valid long-term determinant, as well as the apparent efficacyof aminoguanidine as a therapeutic agent.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

What is claimed is:
 1. A method for detecting the presence of advancedglycosylation endproducts (AGEs) in a biological sample comprising thesteps of:(a) contacting in vitro said biological sample with anantibody, wherein said antibody is an anti-AGE antibody which reactswith an immunological epitope common to AGEs, but which does not reactwith the following model AGEs:2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole(FFI), 1-alkyl-2-formyl-3,4-diglycosyl pyrrole (AFGP),5-hydroxymethyl-1-alkylpyrrole-2-carbaldehyde (pyrraline), andpentosidine, wherein reactivity is detected in a competitive solid phaseassay format, wherein bovine serum albumin (BSA)-AGE obtained byincubation of BSA with glucose is adsorbed to said solid phase, and saidmodel AGE is tested as the inhibitor of binding of said antibody to saidBSA-AGE; (b) allowing the formation of reaction complexes which comprisesaid antibody and said AGEs; and (c) detecting the formation of saidreaction complexes in said biological sample; wherein detection of saidreaction complexes indicates the presence of AGEs in said biologicalsample.
 2. The method of claim 1 wherein said anti-AGE antibody is boundto a solid phase support.
 3. The method of claim 1 wherein an isolatedAGE is bound to a solid phase support, which isolated AGE is not2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI),1-alkyl-2-formyl-3,4-diglycosyl pyrrole (AFGP),5-hydroxymethyl-1-alkylpyrrole-2-carbaldehyde (pyrraline), orpentosidine, wherein said anti-AGE antibody is labelled and forms areaction complex with said solid phase support AGE, further comprisingcontacting said biological sample in step (a) with said solid phasesupport AGE and said labelled antibody, removing any substance not partof said reaction complex comprising said labelled antibody and saidsolid phase support AGE formed in step (b) prior to step (c), whereinthe formation of said reaction complexes comprising said antibody andsaid AGEs in said sample is detected by observing a decrease in theamount of labelled antibody in said reaction complexes with said solidphase support AGE compared to a control sample lacking AGEs.
 4. Themethod of claim 1 wherein said biological sample is selected from thegroup consisting of blood; plasma; urine; cerebrospinal fluid; lymphaticfluid; tissue; plant matter; edible animal matter; a product of thereaction between protein and a sugar; a product of the reaction betweenDNA and a sugar, and mixtures thereof.
 5. The method of claim 1 whereinsaid AGE in said biological sample is selected from the group consistingof Hb-AGE, mammalian serum albumin-AGE, serum AGE-proteins, serumAGE-peptides, urinary AGE-peptides, and combinations thereof.
 6. Amethod for determining the level of AGEs in a biological samplecomprising quantifying the formation of said reaction complexes bymeasuring the extent of binding detected in said biological sampleaccording to the method of claim 1 wherein the quantity of said reactioncomplexes corresponds to the level of AGEs in said biological sample. 7.A method for monitoring time-integrated blood sugar levels in a patientcomprising determining the level of AGEs in a biological samplecomprising quantifying the formation of said reaction complexes bymeasuring the extent of binding detected in said biological sampleaccording to the method of claim 1, wherein the quantity of saidreaction complexes corresponds to the level of AGEs in said biologicalsample.
 8. A method for diagnosing the presence of a disease or disorderassociated with elevated AGE levels in a mammalian subjectcomprising:(a) determining the level of AGEs in a biological sample byquantifying the formation of said reaction complexes by measuring theextent of binding detected in said biological sample according to themethod of claim 1, wherein the quantity of said reaction complexescorresponds to the level of AGEs in said biological sample; and (b)comparing the level determined in step (a) to a level of AGEs present ina normal mammalian subject of the same species;wherein detection of ahigher level of AGEs as compared to a normal level is indicative of thepresence of a disease or disorder associated with elevated levels ofAGEs.
 9. The method of claim 8 wherein the disease or disorder isselected from the group consisting of diabetes, glycemia, urinarydiseases and dysfunctions, chronological aging, the effects of proteinaging, and combinations thereof.
 10. A method for monitoring the courseof a disease associated with elevated AGE levels in a mammalian subjectcomprising determining the level of AGEs in a series of biologicalsamples obtained at different time points from a mammalian subject byquantifying the formation of said reaction complexes by measuring theextent of binding detected in each of said biological samples accordingto the method of claim 1, wherein the quantity of said reactioncomplexes corresponds to the level of AGEs in each of said biologicalsamples, wherein an increase in the level of AGEs over time indicatesprogression of said disease, and wherein a decrease in the level of AGEsover time indicates regression of said disease.
 11. A method formonitoring therapeutic treatment of a disease associated with elevatedAGE levels in a mammalian subject comprising determining the levels ofAGEs in a series of biological samples obtained at different time pointsfrom a mammalian subject undergoing a therapeutic treatment for adisease associated with elevated AGE levels by quantifying the formationof said reaction complexes by measuring the extent of binding detectedin each of said biological samples according to the method of claim 1,wherein the quantity of said reaction complexes corresponds to the levelof AGEs in each of said biological samples, wherein a decrease in thelevel of AGEs over time indicates an effective therapeutic outcome. 12.A method for the long term monitoring of blood sugar in a mammalcomprising:obtaining a sample of serum protein from a mammal;determining the level of advanced glycosylation endproduct serum proteinby quantifying the formation of said reaction complexes by measuring theextent of binding detected in said biological sample according to themethod of claim 1, wherein the quantity of said reaction complexescorresponds to the level of AGEs in said biological sample; andcomparing the level of advanced glycosylated endproduct serum protein toa level in said mammal obtained at an earlier time, wherein an increaseindicates an increase in blood sugar, and a decrease indicates adecrease in blood sugar.
 13. A test kit for measuring the presence ofAGEs in a biological sample in vitro, comprising:(a) an anti-AGEantibody reactive with AGEs, but does not react with the following modelAGEs:2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI),1-alkyl-2-formyl-3,4-diglycosyl pyrrole (AFGP),5-hydroxymethyl-1-alkylpyrrole-2-carbaldehyde (pyrraline), andpentosidine, wherein reactivity is detected in a competitive solid phaseassay format, wherein bovine serum albumin (BSA)-AGE obtained byincubation of BSA with glucose is adsorbed to said solid phase, and saidmodel AGE is tested as the inhibitor of binding of said antibody to saidBSA-AGE; (b) a labelled reagent capable of forming a complex with an AGEor with said anti-AGE antibody; and (c) directions for use of said kit.14. The test kit of claim 13 wherein said anti-AGE antibody is bound toa solid phase.
 15. The test kit of claim 13 further comprising anisolated AGE as a standard, excluding the following modelAGEs:2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI),1-alkyl-2-formyl-3,4-diglycosyl pyrrole (AFGP),5-hydroxymethyl-1-alkylpyrrole-2-carbaldehyde (pyrraline), andpentosidine.
 16. The test kit according to claim 15 wherein the isolatedAGE is BSA-AGE.